Comestibles sweetened with alpha amino acid dihydrochalcones

ABSTRACT

Dihydrochalcones of the formula   &lt;IMAGE&gt;   are disclosed wherein X is H or OH and R is a lower alkyl. These materials are useful as sweeteners for edibles. A process for preparing these compounds using a novel intermediate is disclosed as are acid and base neutralization products of the subject dihydrochalcones.

This is a division and Continuation-in-Part of copending applicationSer. No. 19,054, filed on Mar. 9, 1979.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention concerns synthetic sweeteners. More particularly, itconcerns a new group of dihydrochalcone compounds and their use assweeteners for edible compositions such as foodstuffs.

2. Discussion of the Prior Art

Dihydrochalcones are compounds having a ##STR2## basic structure.Various examples of dihydrochalcones have been known since the 1930s.The members of this class of dihydrochalcones vary from one another bythe nature and placement of substituents on the aromatic rings.

In 1963 it was discovered that some, but by no means all, of thedihydrochalcones are sweet (Horowitz and Gentili, U.S. Pat. No.3,087,821, issued Apr. 30, 1963). The earliest examples of sweetdihydrochalcones were derived from naturally occurring flavanones havingbulky saccharide residues attached at position 4. More recently, severalsweet dihydrochalcones having smaller and simpler substituents at their4 position have been disclosed. Such disclosures include Rizzi, U.S.Pat. No. 3,855,301, issued Dec. 17, 1974; Rizzi, U.S. Pat. No.3,751,270, issued Aug. 7, 1973; Farkus et al., U.S. Pat. No. 3,956,375,issued May 11, 1976; Crosby et al., U.S. Pat. No. 3,974,299, issued Aug.10, 1976; Crosby et al., U.S. Pat. No. 3,976,790, issued Aug. 24, 1976;Crosby et al., U.S. Pat. No. 4,055,678, issued Oct. 25, 1977 and U.S.Ser. No. 964,211, filed by Crosby et al. on Dec. 1, 1978.

Among the prior art references, the last mentioned patents andapplication of Crosby et al. disclose compounds which are considered tobe structurally closest to the present materials. The prior artcompounds have substituents in the 2, 6 and 4' positions which are thesame as those of the present compounds. Importantly, however, thesereferences do not show or suggest the alpha amino acid 4 positionsubstituent which the present compositions require. All employ groupswhich are chemically far different from the present alpha amino acid.

If there is one fact that may be derived from the prior art, it is thatno underlying taste-structure relationship has been developed or provedfor the dihydrochalcones. Changes which are chemically minor on theirface may have a major effect on taste properties. The taste/structurerelationship still is largely empirical and not predictable.

STATEMENT OF THE INVENTION

The present invention is a new class of sweet dihydrochalcones. Thesematerials are classifiable as4-(3-amino-3-carboxypropoxy)-dihydrochalcones. They are representedstructurally in their most common ionic state as shown in GeneralFormula I. ##STR3## wherein R is a lower saturated alkyl of from 1 to 4carbons inclusive, and X is hydrogen or hydroxy. These materials may benamed 2,3'6-trihydroxy and2,3'-dihydroxy-4-(3-amino-3-carboxypropoxy)-4'-alkoxydihydrochalcones.

These compounds contain several ionizable protons and a cationic groupsubject to protonation. This means that at pHs below about 3 or aboveabout 8, additional ionic forms of the dihydrochalcones are present insignificant amounts.

At low pHs the carboxyl group protonates to give the compound shown inGeneral Formula II. ##STR4## wherein X and R are previously defined andZ⁻ is a physiologically acceptable conjugate base of a strong acid.

Depending upon the identity of X, the compounds of Formula I have 2 or 3labile hydrogens which can be ionized at high pHs. One hydrogen can beionized from the amine at pHs above about 8. At about the same pH ahydrogen can be ionized from the 6 position hydroxyl (i.e., if X is OH).The 3' hydroxyl can give up a hydrogen as well, but this hydrogen is oneor two orders of magnitude less acidic than the first two. If X ishydrogen, instead of hydroxyl, obviously, the molecule can give up atmost two protons.

The high pH forms of the compound may be depicted structurally as shownin General Formula III. ##STR5## wherein R is as previously defined, M⁺is a physiologically acceptable cation; X is hydrogen or oxy, and y andz are each positive numbers, the sum of which totals 2 when X ishydrogen or 3 when X is oxy.

Each of these forms of the compounds of this invention constitutes anaspect of the invention as does the use of these materials to impartsweet flavors to foods, beverages, medicaments and other comestibles.

These compounds may be formed by substituting the 7 position of theflavanones shown in General Formula IV. ##STR6## with the electrophilicsubstitution reagent methyl 2-(N-carbobenzyloxy)amino-4-bromobutyrate orthe like and thereafter opening the flavanone and hydrogenating to thedihydrochalcone.

DETAILED DESCRIPTION OF THE INVENTION The Compounds

The compounds of the present invention in their most common state havethe structure shown in General Formula I. In that Formula R is an alkyl,more particularly a 1, 2, 3, or 4 carbon alkyl that preferably islinear, i.e., methyl, ethyl n-propyl or n-butyl. Methyl is the mostpreferred R.

X is either hydrogen or hydroxy, with hydroxy being preferred. M⁺ is aphysiologically acceptable cation. As used herein, a "physiologicallyacceptable cation" is defined to include ammonium and the cations of thethird and fourth period metals which are nontoxic, especially Na(I),K(I), Mg(II), Ca(II), Al(III) and Zn(II). Preferred cations are thecations of the third and fourth period group I and II metals, i.e.,Na(I), K(I), Mg(II), and Ca(II), with K being the most preferred cation.In structural formulae of this specification, the divalent calciumcation will be shown as 1/2 Ca⁺⁺ to indicate a charge balance with themonovalent carboxy group. In actual practice, of course, the Ca⁺⁺ isassociated with two monovalent dihydrochalcone groups. Mixtures ofcations may be used as well.

Z⁻ is a physiologically acceptable conjugate base of a strong acid suchas the mineral acids. As used herein, the term "strong acid" refers toan acid which is at least 90% ionized in pH 3 water while a"physiologically acceptable conjugate base" is a base which is nontoxic.Representative physiologically acceptable conjugate bases of strongacids include Cl⁻, HSO₄ ⁻, SO₄ ⁻⁻ and H₂ PO₄ ⁻.

While it will be appreciated that Formulae I, II and III represent thesame compound, but in different ionic states, the form shown in FormulaI is the most common and thus generally preferred.

PREPARATION OF THE COMPOUNDS

The compounds of General Formula I are conveniently formed, in a generalsense, by substituting the 7 position of the flavanones shown in GeneralFormula IV. ##STR7## with the electrophilic substitution reagent, methyl2-(N-carbobenzyloxy)amino-4-bromobutyrate, and thereafter removingprotecting groups and converting the flavanone to the desireddihydrochalcone. The flavanones include hesperetin and its X equalshydrogen and R equals C₂ H₅,C₃ H₇ or C₄ H₉ equivalents. Methyl2-(N-carbobenzyloxy)amino-4-bromobutyrate has the formula ##STR8## Itspreparation and a representative preparation of the subjectdihydrochalcones are given in the Examples.

The general preparative scheme for the dihydrochalcones may be shown asfollows: ##STR9##

The addition of the bromobutyrate to the flavanone 7 position (Step A,above) is carried out as follows. The bromobutyrate and the flavanoneare combined in a liquid phase polar aprotic reaction medium. Suitablemedia include N,N-dimethylformamide (DMF), N-methylpyrrolidinone (NMP),dimethylsulfoxide, hexamethylphosphoramide, and the like with NMPgenerally being preferred. The molar amounts of flavanone andbromobutyrate are about equal with 0.2 to 1.0, preferably 0.5 to 1.0 andmost preferably 0.8 to 1.0 equivalents of flavanone per mole ofbromobutyrate being employed.

An acid acceptor, such as an alkali metal carbonate, bicarbonate orhydroxide, and preferably Na₂ CO₃ or K₂ CO₃, is present during thebromobutyrate substitution reaction. This material is generally presentin a molar amount about equal to the moles of flavanone--i.e., 1-1.5equivalents, basis flavanone. The reaction is carried out under moderateconditions such as temperatures of from 10° C. to 60° C. for times offrom 5 to 70 hours. Time, of course, is inversely proportional totemperature. The mixture is generally stirred and blanketed with aninert gas atmosphere. The bromobutyrate substitution product may berecovered and purified by simple art-known techniques such asextraction, crystallization, evaporation of solvent, and the like.

The cleavage and hydrogenation of the flavanones to the dihydrochalcones(Step B, above) is carried out with molecular hydrogen and a suitablecatalyst. Mild conditions, such as a gross excess of hydrogen (forexample 10 to 100 psi), dilute aqueous base such as 1 to 8 molar,preferably 2 to 6 molar alkali metal hydroxide, particularly KOH or NaOHand a noble metal catalyst such as palladium or platinum, (preferablypalladium), preferably supported such as upon charcoal or the like.Times of from a few hours to about 30 hours, with temperatures of fromroom temperature (20° C.) to say 35° C. may be employed. As earliernoted, more strenuous conditions may be employed, if desired. Theseconditions also serve to remove the protecting groups on the amine andcarboxyl groups.

Following flavanone ring cleavage, hydrogenation, and protecting groupremoval, the product is recovered, such as by filtration to removecatalyst, evaporation to dryness and chromatography, such as by liquidchromatography or other equivalent chromatographic techniques, or bycrystallization, especially in pH range of from 4 to 7. Crystallizationis the preferred recovery technique.

The flavanones employed as starting materials in this synthesis includehesperetin and its X and R substituted equivalents. Hesperetin (X=OH,R=CH₃) is available commercially. The other flavanones are less commonand generally must be prepared. One preparative route for theseflavanones involves condensation of an appropriately protectedhydroxyacetophenone with an appropriately protected3-hydroxy-4-alkoxybenzaldehyde in the presence of base to give achalcone which is then converted to the desired flavanone by treatmentwith strong acid. This route may be shown as follows: ##STR10##

As will be now shown, these steps can be carried out with processconditions and reagents known to those skilled in the art. The protectedhydroxyacetophenone derivatives, such as2-hydroxy-4,6-dibenzyloxyacetophenone and2-hydroxy-4-benzyloxyacetophenone, are prepared from the requisitecommercially available hydroxyacetophenones by treatment with a reagentsuch as a benzyl halide, particularly benzyl bromide or iodide, orchloride (1.00-1.25 equivalent based upon the number of hydroxyl groupsto be reacted) at 25°-80° C. in polar aprotic liquid phase media.Suitable media include N,N-dimethylformamide (DMF), NMP, DMSO,hexamethylphosphoramide, and the like. An acid acceptor, such as a metalbicarbonate, carbonate, or hydroxide, especially an alkali metal such asK⁺ of a bicarbonate, carbonate or hydroxide, is also added to thereaction mixture in an amount of from 0.8 to 1.5 equivalents per mole ofhydroxyl group being protected. Generally, long reaction times, such asat least 12 hours, are employed with these mild conditions. The mostpreferred method for preparing the protected hydroxyacetophenonesinvolves the use of benzyl chloride (1.1 equivalent) and K₂ CO₃ (1.0equivalent) in DMF at 25°-40° C. Under these conditions the reactionsare complete within 3-4 days, with product isolation being carried outby means of a standard aqueous workup.

The protected 4-alkoxy-3-hydroxybenzaldehydes, needed for condensationwith the protected hydroxyacetophenones, are prepared by a two-stepprocess from 3,4-dihydroxybenzaldehyde (protocatechualdehyde;commercially available). The first step, which is the preparation of theintermediate 4-alkoxy-3-hydroxybenzaldehydes, involves the treatment ofthe 3,4-dihydroxybenzaldehyde with 1.0-1.1 molar equivalents of a 1-3carbon alkyl halide (especially iodide) in a polar aprotic solvent, suchas DMF, at room temperature or slightly above (15°-40° C.). An acidacceptor, such as an alkali metal carbonate, bicarbonate or hydroxideand preferably K₂ CO₃, in a molar amount about equal to the moles ofalkyl halide is required for this reaction. When carried out under thesemild conditions, the hydroxyl group at the 4-position, being somewhatmore reactive than the hydroxyl group at the 3-position, is alkylatedalmost exclusively. Protection of the remaining hydroxyl is theneffected preferably by benzylation such as at 25°-50° C. with eitherbenzyl chloride or benzyl bromide in DMF or a similar solvent containing1.0-1.2 molar equivalents of K₂ CO₃. This completes the preparation ofthe 4-alkoxy-3-benzyloxybenzaldehydes or their otherwise protectedequivalents.

The aldol condensation of the protected hydroxyacetophenones with the4-alkoxybenzaldehydes, to afford a chalcone, is best carried out with aslight molar excess (preferably 1.1 to 1.5 molar equivalents, basisacetophenone) of benzaldehyde in a lower alkanol (methanol, ethanol,isopropanol) at room temperature to 75° C. A large excess (10-20 molarequivalents) of a strong base, such as NaOH, KOH, NaOEt, or t-BuOK, isneeded in order for this reaction to proceed at a reasonable rate. Thepreferred method for conducting this aldol condensation is to utilizeabout 1.25 molar equivalents of the benzaldehyde and about 15 molarequivalents of 60% aqueous KOH in absolute ethanol (1.0 ml/mmol ofacetophenone) at 20°-30° C. Under these conditions, the condensation iscomplete within 72 hours. The chalcone products may be isolated, afterneutralization of the reaction mixture, by either a standard aqueousworkup or by evaporating the reaction mixture to dryness and thenextracting the product from the salts. Purification is carried out byrecrystallization, with ethanol being the preferred solvent.

The chalcones, when protected as preferred with benzyl groups, undergodebenzylation with concomitant cyclization to the flavanones upontreatment with excess very strong mineral acid. Aqueous HI or HBr (10-20molar equivalents) in glacial acid (20-60 ml/mmol of chalcone) arepreferred acids and are employed at mildly elevated temperatures(30°-100° C.). In general, these reactions proceed rather poorly withother mineral acids, such as HCl, H₂ SO₄, or HClO₄. The productflavanones are isolated, as a mixture with the resulting benzyl halidecoproduct, by a standard aqueous workup. Purification is bestaccomplished by chromatographic techniques, such as thick layerchromatography or column chromatography. All of these reactions may beadvantageously carried out with stirring and under an inert gasatmosphere.

USE OF THE DIHYDROCHALCONE PRODUCTS

The dihydrochalcone products of this invention are sweet. The sweetnessis intense--300-500 times that of sucrose. The sweetness ispure--greater than 85% of the flavor is sweet. The timing of thesweetness is sucrose-like. The taste sensation arrives quickly anddeparts without substantial linger in an excellent mimic or sucrose.

The dihydrochalcone products of this invention are sweet. They may beused as nonsucrose sweeteners for edibles (i.e., comestibles) such asfoods, medicaments and beverages. In this use they may be admixed suchas by dissolving or dry mixing with the edible in an amount about1/100-1/1000 that which would be appropriate for sucrose. Thus, amountsof from about 0.2 to 0.005% by weight (basis edibles) may be employed.

One difficulty should be noted in the usefulness of these materials. Thecompounds, especially in a high state of purity, are very prone tocrystallize out of solution, especially out of cold, weakly acidic (pH2-7) solely aqueous solutions.

One may, however, minimize the impact of this property in a number ofways. For example, one may use the salt forms, especially the mono anddi-alkali and alkaline earth salts (especially potassium or sodium) andammonium salts. Such forms offer improved solubility such as for drytable-top sweetener use. Another approach is to use the compounds withan organic cosolvent or solubilizer. Suitable materials are organicliquids such as glycerol and propylene glycol as well as aqueoussolutions of materials such as sorbitol (70% aqueous solution) anderythritol aqueous solution. These organic solubilizers may be addedadvantageously in the range of from 0.1% by weight to 5% by weight(preferably 0.2% by weight to 3% by weight) basis total solution.Additionally, many consumable systems contain suspended or dissolvedmaterials which beneficially interfere with crystallization and thusimprove solubility. For example, brewed coffee contains solids and oilswhich enhance solubility. Yet another approach to minimizing the problemof marginal solubility, as it might appear in soft drink beveragesystems, is to incorporate the sweetener into the highly acidic beverageconcentrate (contains phosphoric acid) prior to diluting the concentraterather than after dilution.

The present invention will be further shown by the following examples.These are intended to exemplify the invention and are not to beconstrued as limiting its scope.

EXAMPLES Preparation of Precursors I. Methyl2-(N-carbobenzyloxy)amino-4-bromobutyrate

This compound is prepared by a modification of the procedure used byNollet, Huting and Pandit to prepare tertiary butyl2-(N-carbobenzyloxy)amino-4-bromobutyrate. ##STR11## To 3.64 g (20.0mmoles) of 2-aminobutyrolactone hydrobromide is added 182 ml of glacialacetic in a 500 ml three-necked flask equipped with a fritted glass gasbubbler, calcium chloride drying tube, and magnetic stirrer. Hydrogenbromide gas is then vigorously bubbled into the resultant suspensionwhile stirring at ambient temperature. After three hours, all of thestarting material has gone into solution and hydrogen bromide additionis discontinued. The reaction mixture is stirred at ambient temperatureovernight and then is concentrated to dryness at reduced pressureyielding 5.2 g of crude 2-amino-4-bromobutyric acid hydrobromide as awhite solid. This crude acid is dissolved in 25 mls absolute methanolafter which hydrogen bromide gas is bubbled in for one minute.Monitoring reaction progress by NMR (BrCH₂ --:CH₃ --O--integration)indicates negligible reaction after stirring overnight at roomtemperature. After refluxing for 72 hours the reaction is judged to becomplete. The reaction mixture is then concentrated to dryness atreduced pressure yielding crude methyl 2-amino-4-bromobutyratehydrobromide as a colorless oil. To a solution of this crude ester in 25ml water is added 3.70 g (44.0 mmoles) of sodium bicarbonate whilestirring vigorously in an ice bath. Immediately, 3.8 mls (4.1 g; 24mmoles) of benzylchloroformate is added dropwise over ca. two minutes.After ten minutes, the ice bath is removed, allowing the reactionmixture to warm to room temperature over 30 minutes. It is then pouredinto a separatory funnel and extracted with ether (3×25 ml), thecombined portions of which are washed with saturated sodium bicarbonate(3×25 ml), dried over magnesium sulfate and concentrated, yielding 5.44g of an oily white solid. Recrystallization from hexane-ether yields1.96 g of methyl-2-(N-carbobenzyloxy)-amino-4-bromo-butyrate (A) aswhite clusters having mp 87°-9°. This represents a 30% overall yieldfrom 2-amino-butyrolactone hydrobromide. Tlc (Silica Gel F-254; CHCl₃)shows one spot having R_(f) =0.15.

IR: λ_(Max) ^(KBr) 2.95 (N-H), 3.32, 3.40, 5.83, (C═O), 6.59, 6.97,7.46, 8.20, 9.02, 9.16, 10.90, 12.83, 13.36, 14.27μ.

NMR: δ_(CDCl).sbsb.3^(TMS) 1.99-2.56 (2H multiplet, Br--C--CH₂--C--N--), 3.40 (2H triplet, J=7.2 Hz, Br--CH₂ --, 3.76 (3H singlet,O--CH₃), 5.10 (2H singlet, Ar--O--CH₂), 5.30 (1H broad abs, N-H), 7.36(5H singlet, aromatic H).

ANAL: Calc. for C₁₃ H₁₆ BrNO₄ : C, 47.28; H, 4.88; Found: C, 47.98; H,4.95.

II. Flavanones by Condensing Aldehydes and Acetophenones A. Preparationof Unprotected Aldehyde Reactants

(1) A solution-suspension of 2.76 g (20.0 mmoles) of3,4-dihydroxybenzaldehyde and 2.76 g (20.0 mmoles) of anhydrouspotassium carbonate and 3.45 g (22.0 mmoles) of ethyl iodide is preparedin 15 ml of dry DMF and stirred under argon for 24 hours at roomtemperature. The reaction mixture is poured into 50 ml dilute HCl,saturated with sodium chloride and extracted thrice with diethyl ether.The ether extracts are washed with water, and brine, dried andconcentrated to yield the ethoxyaldehyde as dark crystals. ##STR12##

(2) The reaction is repeated using 3.74 g (22.0 mmoles) of n-propyliodide in place of ethyl iodide to yield the propoxyaldehyde ##STR13##

(3) The reaction could be repeated using 3.12 g (22.0 mmoles) of methyliodide in place of ethyl iodide to yield the methoxyaldehyde ##STR14##Alternatively, this material, isovanillin, is readily available,commercially.

(4) The reaction is repeated using 4.36 g (22.0 mmoles) of n-butyliodide in place of ethyl iodide to yield the butoxyaldehyde ##STR15##

B. Preparation of 4-Alkoxy-3-benzyloxybenzaldehyde ##STR16##

4-Alkoxy-3-hydroxybenzaldehyde (1.0 equiv.), benzyl chloride (1.2equiv.), and K₂ CO₃ (1.2 equiv.) are stirred in anhydrous DMF at 35° C.for 72 hours. The reaction mixture is poured into dilute HCl, extractedthrice with ether and the combined extracts washed thoroughly with H₂ O,dilute aqueous KOH (until the ether solution is free of unreactedhydroxybenzaldehyde as determined by TLC), H₂ O again, and finallybrine. Evaporation affords crude 4-alkoxy-3-benzyloxybenzaldehyde whichis generally suitable for use, as is, in the condensation reaction.Additional purification may be achieved by silica gel columnchromatography.

C. Preparation of Protected Acetophenones (1) Preparation of2-Hydroxy-4,6-dibenzyloxyacetophenone ##STR17##

2,4,6-Trihydroxyacetophenone (16.8 g, 0.10 mol, Aldrich ChemicalCompany) and benzyl chloride (27.8 g, 2.00 mol) are dissolved in 200 mlof dry DMF and the solution is thoroughly purged with argon. The mixtureis treated with 27.6 g (0.20 mol) of K₂ CO₃ and stirred at 35° C. for 84hours. The reaction is poured into ether (1200 ml) and resulting mixturewashed with H₂ O (6×500 ml), 5% aqueous KOH solution (3×500 ml), H₂ O(1×500 ml), and saturated NaCl solution (1×250 ml). After drying overMgSO₄, the ethereal solution is evaporated to afford 27.4 g of crudeproduct as an off-white granular solid. Trituration of the crude productwith ether (100 ml), followed by filtration and drying in vacuo provides13.5 g (38.8%) of 2-hydroxy-4,6-dibenzyloxyacetophenone as a whitesolid, mp 101°-102° C., i.e., ##STR18## wherein R' is benzyl.

The product is homogeneous by silica gel TLC (CHCl₃ elution) and theassigned structure is verified by both NMR and elemental analysis.

(2) Preparation of 2-hydroxy-4-benzyloxyacetophenone

The reaction of (1) above is repeated using 1.1 molar equivalents ofbenzyl chloride, 1.0 molar equivalent of K₂ CO₃, and substituting forthe above acetophenone, 2,4-dihydroxyacetophenone ##STR19##

D. Preparation of 2-Hydroxy-3',4,6-tribenzyloxy-4'-alkoxychalcone##STR20##

Twelve mls of warm 60% aqueous KOH is added rapidly to a solution of2.79 g (8.00 mmoles) 2-hydroxy-4,6-dibenzyloxyacetophenone and 1.00equivalent (8.00 mmoles) 3-benzyloxy-4-alkoxybenzaldehyde in a mixtureof 16 mls absolute ethanol and 16 mls tetrahydrofuran while stirringvigorously under argon. After 16 hours, the reaction mixture is pouredinto an excess of ice-cold HCl and the precipitated yellow chalconeisolated by filtration. After washing with H₂ O and air drying, thechalcone is recrystallized from ethanol-ethylacetate to yield the purechalcone as tiny yellow needles (65-90%). Product identity and purityare determined by TLC [Hexane-Ethyl Acetate (1:1)], proton nmr andelemental analysis.

E. Preparation of 3',5,7-Trihydroxy-4'-alkoxyflavanone ##STR21##

A 1.0 mmol sample of 2-hydroxy-3',4,6-tribenzyloxy-4'-alkoxychalcone isdissolved in 40 ml of glacial acetic acid at 60° C. and treated with 2ml of 48% aqueous HBr. The yellow solution becomes deep reddish-orangeupon addition of the acid. After stirring 24 hours at this temperature,the reaction mixture is poured into H₂ O (200 ml) and the resultingaqueous mixture extracted with an equal volume of ethyl acetate. Theorganic extract is washed with H₂ O (2×100 ml), 5% aqueous NaHCO₃solution (2×100 ml), H₂ O (1×100 ml), saturated aqueous NaCl solution(1×50 ml), and dried over MgSO₄. Evaporation affords the crude flavanoneadmixed with three equivalents of benzyl bromide.

Silica gel column chromatography (elution with ethyl acetate-hexane,(1:1) affords flavanone (30-60%) as an off-white crystalline solid,which may be further purified by recrystallization. Product identity andhomogeneity are determined by silica gel TLC (ethyl acetate-hexane,1:1), proton NMR, and elemental analysis.

F. The coupling, exemplified by Parts D. and E., is repeated six moretimes varying the aldehyde among the four materials of Part A. of thispreparation and the two acetophenones of Part B. so, with the materialsof D. and E., as to yield the eight possible flavanones of GeneralFormula II which can result when X is H or OH and R is CH₃, C₂ H₅, C₃ H₇or C₄ H₉.

EXAMPLE I Preparation of Dihydrochalcone Wherein X is OH and R is CH₃ A.3,5-Dihydroxy-4'-methoxy-7-(3-(N-carbobenzyloxy)amino-3-carbomethoxy-propoxy)-flavanone##STR22##

Seven mils of dry dimethylformamide are added to a mixture of 302 mg(1.00 mmole) hesperetin, 152 mg (1.10 mmoles) anhydrous potassiumcarbonate, and 330 mg (1.00 mmole) of methyl2-(N-carbobenzyloxy)-amino-4-bromo-butyrate, made as above. Theresulting mixture is stirred vigorously under an argon temperature at35° overnight after which it is diluted with 40 mls water, acidified topH 5 with 1 N HCl and extracted with ethyl acetate (3×25 ml), thecombined portions of which are washed with water (6×20 ml), brine (1×20ml), dried over magnesium sulfate and concentrated yielding 585 mg of anoff-white foam. Purification by preparative Tlc (Silica Gel PF-254; CH₂Cl₂ CH₃ OH [98:2]) yields 368 mg (67%) of the pure alkylation product"B" as a colorless oil. Tlc (Silica Gel F-254; CH₂ Cl₂ --CH₃ OH [98:2])showed one spot having R_(f) =0.22.

IR: λ_(max) ^(film) 2.96 (O--H), 3.24, 3.36, 3.44, 5.81 (ester,carbamate C═O), 6.09 (ketone C═O), 6.35, 6.54, 6.62, 6.85, 6.96, 7.30,7.48, 7.73, 7.89, 8.06, 8.38, 8.61, 8.85, 9.19, 9.40, 9.59, 9.75, 11.52,12.43, 13.13, 13.48, 14.35μ.

NMR: δ_(CDCl).sbsb.3^(TMS) 2.00-2.53 (2H multiplet, --O--C--CH₂--C--N--), 3.72 (3H singlet, --COOCH₃), 3.87 (3H singlet, Ar'--O--CH₃),4.00 (2H triplet, J-7.4 Hz, --O--CH₂ --C--C--N--), 4.36-4.70 (1Hmultiplet, --CH--N--), 5.10 (2H singlet, PhCH₂ O--), 5.10-5.39 (1Hmultiplet, Ar--O--CH--Ar'), 6.00 (2H singlet, Ar aromatic H), 6.76-7.07(3H multiplet, Ar' aromatic H), 7.37 (5H singlet, benzyl aromatic H),11.98 (1H singlet, Ar--O--H).

B.2,3'6-Trihydroxy-4-(3-amino-3-carboxy-propoxy)-4'-methoxy-dihydrochalcone##STR23##

To a solution of 368 mg (0.67 mmole) of flavanone B in 35 mls of 5%potassium hydroxide is added 210 mg of 5% Pd-C while under an argonatmosphere. The resultant reaction mixture is shaken on a Parrhydrogenator at 30 lb hydrogen pressure for 15 hours after which it isfiltered through celite yielding a light green solution. Adjustment ofthe pH to 6 with 10% HCl results in formation of a large amount ofoff-white precipitate. Filtration yields 151 mg (56%) of the pure aminoacid as a white solid having mp 182°-4° (dec). Tlc (cellulose F-254;i-BuOH-HOAc-H₂ O [2:1:1]) shows one spot having R_(f) =0.73. HPLC on aWaters Associates instrument (30 cm C-18 on μ-Bondapak column elutingwith 10-100% methanol in 0.03 M NaH₂ PO₄ buffer (pH=4.8) gradient andemploying a Schoeffels UV detector [286 nm] ) shows one peak havingRT=12.2 minutes.

IR: λ_(Max) ^(KBr) 2.90 (O--H, N--H), 6.36 (C═O), 6.42 (C═O), 6.62,7.00, 8.37, 8.69, 9.22, 11.27, 12.3μ.

UV: λ_(Max) ^(MeOH) 226 (ε=1.47×10⁴), 284 (ε=1.45×10⁴) nm.

NMR: δ_(CD).sbsb.3_(OD) ^(TMS) 2.04-2.55 (2H multiplet, O--C--CH₂--C--N), 2.51-2.98 (4H multiplet, Ar--COCH₂ CH₂ --Ar'), 3.82 (3Hsinglet, O--CH₃), 3.90-4.36 (3H multiplet, O--CH₂, C--CH--N), 5.98 (2Hsinglet, Ar aromatic H), 6.60-6.93 (3H multiplet, Ar' aromatic H).

ANAL. Calc. for C₂₀ H₂₃ NO₈ : C, 59.25; H, 5.72; Found: C, 56.12; H,5.89.

As already noted, the dihydrochalcone may exist as the zwitterionprepared in Example I or as the protonated acid or as severaldeprotonated base forms. Example II shows the production of thedeprotonated base forms, while Example III shows production of theprotonated acid form.

EXAMPLE II

A. Preparation of Monopotassium salt of2,3,6-trihydroxy-4-(3-amino-3-carboxypropoxy)-4'-methoxydihydrochalcone.

Twenty-five mls of dioxane (distilled from LiAlH₄) is added to 405 mg(1.00 mmole) of the dihydrochalcone of Example I. After addition of 5mls of distilled water, a homogeneous solution is obtained to which10.00 mls of 0.10 N aqueous KOH (1.00 mmole) is added. The resultantlight yellow solution is then lyophilized to yield the monopotassiumsalt as a solid.

B. The preparation of Part A. is repeated with one change, 2.00 mmolesof KOH are added so as to yield the dipotassium salt.

C. The preparations of Parts A. and B. are repeated with one furtherchange, NaOH is substituted for KOH.

D. A solution of 1.0 equivalent ammonia in methanol is added to asolution of 1.00 eq of the dihydrochalcone of Example I in methanol. Theammonium salt thus formed is then precipitated by addition of ether,isolated by filtration, washed with several portions of ether and driedin a dessicator over KOH pellets.

EXAMPLE III Preparation of Conjugate Acid Salts.

A. 1.00 mmole (405 mg) of the amino acid of Example I is dissolved withheating in 30 mls of IN HCl. On cooling, the hydrochloride salt crystalsare filtered and air-dried to yield 441 mg (100%) of long needles.

B. The preparation of Step A. is repeated substituting 5 N H₂ SO₄ toyield the bisulfate salt as a granular solid.

EXAMPLE IV Preparation of dihydrochalcone wherein X is H, R is CH₃

The preparation of Example I is repeated with one modification. In placeof 1.1 equivalents of hesperetin, an equimolar amount of the flavanoneof General Formula IV, wherein X is H and R is CH₃ and prepared at C(2)above, is employed. This gives rise to the dihydrochalcone of Formula Iwherein X is H and R is CH₃.

EXAMPLE V Preparation of dihydrochalcones wherein X is H and OH and R isC₂ H₅, C₃ H₇ and C₄ H₉

The preparation of Example I is repeated four more times. Each time, anew flavanone prepared at C. above is employed. This results in theformation of the dihydrochalcones in accord with Formula I with thefollowing substituents:

    ______________________________________                                                 R             X                                                      ______________________________________                                                C.sub.2 H.sub.5                                                                             OH                                                              C.sub.2 H.sub.5                                                                             H                                                               C.sub.3 H.sub.7                                                                             OH                                                              C.sub.3 H.sub.7                                                                             H                                                               C.sub.4 H.sub.9                                                                             OH                                                              C.sub.4 H.sub.9                                                                             H                                                       ______________________________________                                    

EXAMPLE VI Preparation of Salts

The salt preparations of Examples II and III are repeated using as thedihydrochalcones those materials made in Examples IV and V.

EXAMPLE VII Use of the Dihydrochalcones and Their Salts as Sweeteners

The compounds of Examples I through VI are employed as sweeteners. Inthis application they are some 350-450 times as intense (on a weightbasis) as sucrose.

In typical applications they may be used as follows.

A 250 mg portion of the material of Example I is placed in a vessel. Tenmls of propylene glycol are then added. The mixture is warmed (45°-50°C.) with gentle stirring. The dihydrochalcone dissolves.

Two ml aliquots are withdrawn and diluted with distilled water, coffee,an unsweetened carbonated cola beverage and an unsweetened cough syrup.In each case a pleasant sucrose-like sweet taste is imparted to thediluent substrate.

A similar experiment is conducted using glycerol as solubilizing agentwith essentially the same results.

The experiment can also be repeated using the dihydrochalcones ofExamples IV and V and the salts of Examples II, III and VI.

What is claimed is:
 1. A sweetened comestible comprising a comestiblematerial in admixture with a concentration of a dihydrochalconerepresented by the formula ##STR24## wherein X is selected from betweenhydrogen and hydroxy and R is an alkyl of from one to four carbon atoms,which is effective to impart sweetness.
 2. The sweetened comestible ofclaim 1 wherein X is hydroxy and R is selected from among methyl, ethyl,n-propyl and n-butyl.
 3. The sweetened comestible of claim 2 wherein thedihydrochalcone is represented by the formula ##STR25##
 4. The sweetenedcomestible of claim 3 wherein said comestible is a beverage.
 5. Thesweetened comestible of claim 3 wherein said comestible is coffee. 6.The sweetened comestible of claim 3 wherein said comestible is tea. 7.The sweetened comestible of claim 4 wherein said comestible is acarbonated beverage.
 8. The sweetened comestible of claim 4 wherein saidcomestible is a noncarbonated beverage.
 9. The sweetened comestible ofclaim 3 wherein said comestible is a toothpaste.
 10. The sweetenedcomestible of claim 3 wherein said comestible is a mouthwash.
 11. Thesweetened comestible of claim 3 wherein said comestible is a chewinggum.